Enzymes have been recognized as biological catalysts since the early 1800's. They have achieved limited commercialization in industrial operations in the last fifty years.
A major deterrent to the large-scale industrial use of conventional water-soluble enzymes has been the high cost involved in one-time use. This obstacle has been removed by immobilization procedures which have resulted in water-soluble enzymes of great stability which can be used repeatedly. The life cycle of an immobilized enzyme is often twenty to forty times greater than experienced with the soluble form.
The earliest observation of enzyme immobilization was in 1916 (Nelson et al. J.A.C.S. 38, 1109 (1916)) deals with enzymes fixed on organic polymers and Mitz [Nature 189, 576 (1961)] covers covalent attachment to cellulose.
More recently, special emphasis has been on carbohydrates as the support for immobilized enzymes. Agarose [Gabel et al., J. Biochem 15, 410 (1970)], Dextran [Axen et al., Nature 210, 367 (1966)], Cellulose [Wheeler et al., Biochem, Biophys., Aeta 191, 187 (1969)], Collodian [Golden et al., Science 150, 758 (1965)], and various derivatized carbohydrates (Enzyme Tech., March 1973) as well as cellulosic fibers [Corno et al., Die Starke 24, 420 (1972)] have been used.
The use of starch gels for immobilizing cholinesterase is known. For use in an analytical procedure Bauman et al., [Anal. Chem. 37, 1378 (1965)] combined the enzyme containing gel with a urethane foam, and used the composition in very thin layers with special precautions to avoid crushing the bed.
Goldstein et al. [Biochem 9, 2323 (1970)] has described enzyme immobilization on a substrate prepared by the condensation of dialdehyde starch with P, P'-diaminodiphenylmethane and subsequent reduction of the Schiff base followed by diazotization and reaction with the protein.
Immobilized enzymes have been used for analysis of various organic compounds including glucose, urea acetylcholine, and uric acid. Industrial utilization has involved DEAE-Sephadex L-amino acidacylase for the continuous resolution of DL-amino acids such as threonine and methionine.
A commercial application of enzymes immobilized on an ion exchange cellulose has been in connection with the partial conversion of starch-derived glucose to fructose, thereby obtaining a syrup of increased sweetness. For this purpose, glucose isomerase enzyme is bound to DEAE-cellulose (or similar cationic cellulose), and the glucose syrup is passed through a series of very thin beds of the isomerase-containing cellulose. See, Schnyder, B. J., "Continuous Isomerization of Glucose to Fructose on a Commercial Basis", Die Starke, 26, 409-412 (1974); and Thompson et al. U.S. Pat. No. 3,788,945, granted Jan. 29, 1974. This technology, as applied commercially by Standard Brands Incorporated and A. E. Staley & Company, involves the use of contact beds of 1 to 5 inches in thickness. This is necessary so that the pressure drop across each bed is small and the compaction of the bed is minimal. But because of the thinness of the beds, in order to avoid the effects of substrate channeling, it is essential to employ a series of such beds. Consequently, fixed-bed columns, such as are used for processes involving ion-exchange resins, cannot be employed. Instead, pressure leaf filters are used. The glucose isomerace bound to the cellulose carried is pumped as an aqueous slurry through the pressure leaf filter in such a manner as to cover each leaf evenly with a thin layer of the cellulose material. As disclosed in U.S. Pat. No. 3,788,945, the depth to width ratio of the beds is preferably limited to from about 0.02 to 0.05.
The plug flow column reactor is one in which the substrate is flowed through a fixed enzyme bed. For large-scale industrial operations, the plug flow reactor is preferred because it results in shorter cycles, lower equipment costs, and a generally more efficient operation. One of the major problems in the use of such reactors is resistance to flow through the immobilized enzyme bed. As the depth of the bed is increased, there is a great tendency for compaction of the bed due to the high pressures which must be employed to pass the substrate solution therethrough. When this occurs, the pressure drop across the bed will increase to such an extent that the pressure necessary to operate may be so high that conventionally constructed equipment cannot be used to contain the bed.
Because of the expense and inconvenience of carrying out glucose isomerization in pressure leaf filters, the corn syrup industry has been actively searching for alternative processes where the glucose isomerase enzyme can be immobilized on a material usable in commercial-size fixed bed columns, which operate as plug flow type reactors. Such column materials must effectively immobilize the enzyme; be chemically and physically stable, resisting disintegration under conditions of use; and being sufficiently porous while minimizing channeling effects, so that there is adequate and uniform contacting but with no excessive pressure drop across the bed. Some microorganisms which produce isomerase contain this enzyme in the cell, and the enzyme is bound therein, or can be bound by a heat treatment. For example, a column material can be prepared for Arthrobacter cells. One process is described in U.S. Pat. No. 3,821,086. Such natural column material shows considerable promise, and provides advantages over the use of shallow beds of cellulose-immobilized isomerase. However, the need is great for a column material of general utility, which can be used for immobilizing soluble isomerase, as well as other soluble enzymes, such as those used for the conversion of starch oligosaccharides to dextrose and maltose. Such a column material could be used to immobilize alpha amylase, glucoamylase, or mixtures thereof, for commercial production of corn starch-derived syrups having D.E.'s from 40 to 97.
In particular, to minimize capitol investment, increase plant capacity and reduce production costs, there has been a manifest need for a column process to produce intermediate D.E. corn syrups, such as the syrups now produced by enzymatic hydrolysis of corn starch to obtain syrups having a D.E. in the range of about 40 to 70. As far as is known, all present commercial processes for producing this type of syrup utilize a final stage batch enzyme treatment in which the soluble enzymes are dissolved in the syrup. This is a one-time enzyme use. Usually, the final stage involves treatment simultaneously with an alpha-amylase and a glucoamylase. Therefore it would be desirable to provide a column material to which a mixture of these enzymes can be chemically bonded and immobilized therein. Such a column material and column apparatus would also have many other applications, and could be used, in general, wherever batch enzyme treatments of substrates with soluble enzymes are now used.